One of the main focuses of the Protein Expression Laboratory is the production of HIV and HIV-related proteins by recombinant DNA methods which are suitable for high resolution structural analysis. The determination of the three-dimensional structure of proteins by the methods of X-ray diffraction and multidimensional NMR requires large quantities (gram amounts) of highly purified and physically homogeneous proteins. With NMR, the determination of protein structures larger than 10 kDa requires protein biosynthetically labeled with combinations of non-radioactive stable isotopes. X-ray crystallography does not have this requirement but the ability to crystallize a protein usually requires that it is highly purified and homogeneous with respect to size and conformation. Structure determination of HIV proteins which are important for the life-cycle of the virus and for its structural integrity are potential targets for rational structure-based drug design. We have supported the structural analyses by NMR of the HIV protease complexed with high affinity inhibitors (D.Torchia, NIDR) and the HIV nef, a protein essential for the pathogenic properties of the virus (Ad Bax, NIDDK). Both studies required the generation of mutants to improve the solubility and stability of the proteins and the development of purification methods to produce large amounts of highly purified proteins. We also developed methods which enabled the proteins to be >90% uniformly labeled with deuterium, required to obtain three dimensional information by NMR. These studies resulted in the first three dimensional structures in solution for these two proteins. The HIV rev is an important regulatory factor required for HIV expression. This RNA binding protein has a strong tendency to self-associate into large polymers or fibers which although suitable for analysis by electron microscopy, preclude analysis by NMR and X-ray crystallography. Various mild detergents have been observed to prevent association and offer the potential for crystallization and NMR analysis. Other HIV proteins including: vif (viral infectivity factor), vpu (involved in viral release from host cells and in the degradation of host CD4) and the capsid proteins, p24, p17 and p7, have been successfully expressed in E.coli. The purification and characterization of these proteins is underway.